FULL PAPER Public Health
Distribution of IS629 and stx genotypes among enterohemorrhagic Escherichia coli
O157 isolates in Yamaguchi Prefecture, Japan, 2004–2013
Mitsuhiro KAMEYAMA1)*, Kiyoshi TOMINAGA1), Junko YABATA1) and Yasuharu NOMURA1)
1)Department
Japan
of Health Science, Yamaguchi Prefectural Institute of Public Health and Environment, 2–5–67 Aoi, Yamaguchi 753–0821,
(Received 22 March 2015/Accepted 24 May 2015/Published online in J-STAGE 6 June 2015)
ABSTRACT. Patterns of insertion sequence (IS)629, norV genotype, and Shiga toxin (Stx) genotype distribution were investigated amongst 203
enterohemorrhagic Escherichia coli O157 isolates collected in Yamaguchi Prefecture, Japan, between 2004 and 2013. A total of 114 IS629
patterns were identified; these were divided into eight IS groups (A–H). Ninety isolates carried an intact norV gene, whereas 113 isolates
carried a norV with a 204-bp deletion. Other than one isolate from IS group G, all isolates with an intact norV belonged to groups A–F,
whereas isolates with a mutant norV belonged to IS groups G and H. Seven stx genotypes were identified, and of those, stx1a/stx2a was
predominant (n=105), followed by stx2c (n=32) and stx2a (n=27). The stx1a/stx2a genotype was associated with the mutant norV isolates,
whereas isolates with an intact norV had the stx2c genotype. Therefore, certain combinations of IS type and stx genotype appear to be more
frequent among O157 clades which may be useful for detection of predominant subtypes in the interest of public health.
KEY WORDS:enterohemorrhagic Escherichia coli, insertion sequence 629, norV, O157, stx genotype
doi: 10.1292/jvms.15-0166; J. Vet. Med. Sci. 77(11): 1437–1441, 2015
Enterohemorrhagic Escherichia coli (EHEC), which produces Shiga toxin (Stx), is a human pathogen that causes
hemorrhagic colitis, encephalopathy and hemolytic uremic
syndrome (HUS) [1, 8]. In Japan, 3,768 cases of EHEC
infection were reported in 2012. Among the EHEC isolates
collected in 2012, the predominant serogroup was O157
(53%), followed by O26 (27%) and O103 (5%) [9].
Insertion sequences (IS), which are small mobile genetic elements, are widely distributed in bacterial genomes.
IS629, a member of the IS3 family of insertion sequences, is
prevalent in the O157 genome; for example, the genome of
O157 strain Sakai contains 98 IS elements, and of these, 23
were identified as IS629 [2, 10]. O157 isolates show diverse
patterns of IS629 insertion, and therefore, IS629 variability
can be used for fingerprinting O157 isolates. Ooka et al. [12]
developed a multiplex PCR tool for screening the distribution of IS629 in the O157 genome.
Several genomic subtyping tools have been developed for
the EHEC O157 serogroup. Kulasekara et al. [6] showed
that two forms of the anaerobic nitric oxide (NO) reductase
gene, norV, are present in O157 isolates: an intact form and
a form with a 204-bp deletion; the intact norV is a putative
determinant of virulence in some O157 isolates, because
NO inhibits Stx2 expression under anaerobic conditions
[15]. In addition, Manning et al. [7] showed that single
nucleotide polymorphisms (SNPs) could be used to classify
*Correspondence to: Kameyama, M., Department of Health Science, Yamaguchi Prefectural Institute of Public Health and Environment, 2–5–67 Aoi, Yamaguchi 753–0821, Japan.
e-mail: [email protected]
©2015 The Japanese Society of Veterinary Science
This is an open-access article distributed under the terms of the Creative
Commons Attribution Non-Commercial No Derivatives (by-nc-nd)
License <http://creativecommons.org/licenses/by-nc-nd/3.0/>.
O157 strains into nine clades and that HUS patients were
significantly more likely to be infected with clade 8 strains.
Further, a lineage-specific polymorphism assay can be used
to classify O157 isolates (lineages I, II and I/II), and lineage
I isolates are more commonly associated with human disease
than lineage II isolates [5, 13, 16, 18].
Some IS elements are thought to play an important role in
the diversification and evolution of bacteria, including those
of EHEC O157 isolates [11, 13]. Yokoyama et al. [17] and
Hirai et al. [3] showed a biased distribution of IS629 among
different lineages or clades of O157 isolates. However, the
isolates examined in these studies were collected from a
limited number of geographical areas, and the distribution
of IS629 among O157 isolates from Yamaguchi Prefecture,
Japan, has not been investigated. In the present study, we
investigated the distribution of IS629 as well as norV types
in EHEC O157 isolates collected in Yamaguchi Prefecture
between 2004 and 2013. We also examined the association
between stx genotype and O157 phylogeny.
MATERIALS AND METHODS
EHEC O157 isolates: A total of 203 EHEC O157 isolates were used in the present study. The isolates were sent
to our laboratory from hospitals and health care centers in
Yamaguchi Prefecture, Japan, between 2004 and 2013,
and originated from epidemiologically unrelated patients
(189 isolates) and asymptomatic carriers (14 isolates). The
clinical symptoms of the patients included watery diarrhea
(n=165), abdominal pain (n=144), bloody diarrhea (n=115),
fever (n=59) and vomiting (n=31). Five patients developed
HUS. All isolates were negative for sorbitol fermentation,
and 192 isolates were serotyped as O157:H7. The remaining isolates were non-motile and were therefore classified
as O157:NM.
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M. KAMEYAMA, K. TOMINAGA, J. YABATA AND Y. NOMURA
DNA preparation: To extract DNA for PCR analysis, individual isolates were cultured on Mueller-Hinton agar (Oxoid, Basingstoke, U.K.), and the DNA was extracted using a
QIAamp DNA Blood mini kit (Qiagen, Hilden, Germany),
according to the manufacturer’s instructions. Prepared DNA
was stored at −20°C until use.
IS629 typing: Multiplex PCR using an O157 IS-printing
system (Toyobo, Osaka, Japan), which can be used to detect
all 32 IS629 loci, was used to type the isolates based on the
number and location of the insertion sequences. The assay
also screens for the presence of the four virulence genes, eae,
hlyA, stx1 and stx2. The resulting banding patterns were analyzed using BioNumerics software v. 7.1 (Applied Maths,
Sint-Martens-Latem, Belgium), which excluded bands corresponding to the 4 virulence genes. A simple matching coefficient and an unweighted pair group method with arithmetic
mean (UPGMA) algorithm were used to generate a dendrogram. For the purposes of this study, isolates with ≥80%
similarity were considered to belong to the same group.
Detection of the intact and deletion variants of norV:
The 2 versions of norV, the intact version and the version
containing a 204-bp deletion, were detected by PCR, as described previously [4].
Detection of isolates belonging to clade 8: We screened
for the presence of clade 8 isolates using mismatch amplification mutation assay (MAMA) PCR, as described previously [4].
stx genotyping: The stx subtypes, stx1a, stx1c, stx1d and
stx2a–stx2g, were detected by PCR, as described previously
[14].
RESULTS
IS629 distribution: A total of 114 IS629 patterns were distinguished amongst the 203 O157 isolates. As shown in Fig.
1, the isolates could be divided into 8 groups (groups A–H).
Group G was predominant (n=103), followed by group C
(n=35). Isolates belonging to each of these 2 groups were
identified in all years of the study (Table 1).
Detection of norV types: Intact norV was detected in 90
isolates (intact norV-type), and norV with the 204-bp deletion was detected in 113 isolates (deletion norV-type). All
isolates belonging to groups A–F, and one isolate from group
G, carried the intact norV (Fig. 1 and Table 1).
Clade 8 isolates: In total, 15 of the 203 O157 isolates
were determined to belong to clade 8. All of these isolates
contained an intact norV gene and were classified into IS
group E.
stx genotypes: The stx1a, stx2a and stx2c subtypes were
detected in 123, 154 and 68 isolates, respectively. The stx1a/
stx2a genotype was predominant (n=105), followed by stx2c
(n=32), stx2a (n=27), stx2a/stx2c (n=21), stx1a/stx2c (n=14)
and stx1a (n=3). Only one isolate carried all three stx subtypes, stx1a, stx2a and stx2c (Table 2).
Among the seven stx genotypes detected, the stx2a
genotype was common in both intact norV-type and deletion norV-type isolates. The stx2c, stx1a/stx2c, stx2a/stx2c
and stx1a/stx2a/stx2c genotypes were common in the intact
norV-type isolates, whereas the stx1a and stx1a/stx2a genotypes were associated with the deletion norV-type isolates.
Of the 15 clade 8 isolates, 7 showed the stx2a genotype,
and 8 showed the stx2a/stx2c genotype.
DISCUSSION
Various IS629 patterns were observed among the O157
isolates collected in Yamaguchi Prefecture, Japan, between
2004 and 2013. The majority of the isolates collected over
this 10-year period belonged to IS groups C and G, although
isolates from 8 different groups were identified. As IS629
is thought to contribute to the diversity of O157 isolates, it
is not surprising that isolates with distinct IS patterns are
distributed in Yamaguchi Prefecture.
Recent studies demonstrated that the distribution of IS629
was biased among O157 clades or lineages [3, 17]. Iyoda et
al. [4] also revealed that O157 clades were associated with
certain norV types, with isolates belonging to clades 4–8
carrying an intact norV, whereas isolates belonging to clades
1–3 contained the deletion type. Our results showed that the
15 clade 8 isolates shared identical or highly similar IS patterns and belonged to a single IS group (group E). Moreover,
with the exception of one isolate, the intact norV-type and
deletion norV-type isolates were clearly divided into distinct
IS groups (IS groups A–F and G–H, respectively). Therefore, our findings, along with those of previous studies [3, 4,
17], indicate that it is rare for isolates belonging to different
clades to share similar IS profiles.
Kulasekara et al. [6] reported that most deletion norVtype isolates contained stx1a, whereas only 10% of intact
norV-type isolates harbored stx1a. In the present study,
95.6% of deletion norV-type isolates and 16.7% of intact
norV-type isolates carried stx1a, in accord with the previous
study. Moreover, all isolates carrying stx2c also carried an
intact norV. A previous study demonstrated that 98.8% of all
stx2c-positive isolates carried an intact norV and belonged to
clades 4–8 [4]. Thus, stx genotypes appear to be associated
with specific O157 clades.
An additional association between stx genotypes and IS
distribution was observed in this study. Among the seven
stx genotypes identified, the predominant genotype, stx1a/
stx2a, was only identified in isolates in IS groups G and H,
whereas the 4 genotypes including the stx2c subtype were
identified in IS groups A–F. Thus, particular stx genotypes
may be associated with O157 isolates with particular IS629
distributions.
Five of the 203 isolates tested in the present study were
obtained from HUS patients. These 5 isolates did not belong
to clade 8 and were genotyped as stx1a/stx2a (n=3, deletion
norV-type) and stx2a/stx2c (n=2, intact norV-type). Similar
results were obtained by Iyoda et al. [4], who found that isolates with these 2 genotypes were significantly more likely
to be associated with HUS patients than with asymptomatic
carriers. Thus, the stx1a/stx2a and stx2a/stx2c genotypes
may be associated with an increased risk of developing
HUS. However, Manning et al. [7] also investigated the association between O157 clades, stx subtypes and HUS and
IS629 DISTRIBUTION AMONG EHEC O157
Fig. 1. Cluster analysis of 203 enterohemorrhagic Escherichia coli O157 isolates based on IS-printing profiles.
Isolates with >80% similarity were considered to belong to the same group. The hatched bar indicates the intact norV,
and the open bar indicates the 204-bp-deletion norV. A “☆” indicates a HUS patient, and “★” indicates an asymptomatic
carrier.
1439
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M. KAMEYAMA, K. TOMINAGA, J. YABATA AND Y. NOMURA
Table 1. Distribution of O157 isolates according to IS groups by year
IS
group
A
B
C
D
E
norVa)
Intact
Intact
Intact
Intact
Intact
F
G
H
Total
Intact
Intact
Deletion
Deletion
Cladeb)
n
clade 8
-
18
1
35
1
3
15
16
1
102
11
145
No. of isolates in each year
2004 2005 2006 2007 2008 2009 2010 2011 2012 2013
5
4
1
1
1
4
1
5
3
2
1
1
1
1
1
1
4
14
15
29
18
8
1
12
10
26
2
2
4
7
5
3
3
1
1
2
3
3
4
1
3
1
9
3
25
13
2
24
8
23
1
8
2
18
3
3
1
9
2
15
1
13
a) Intact or 204-bp-deletion norV gene. b) “−” indicates non-clade 8 isolates.
Table 2. stx genotypes of intact or deletion norV-type isolates
norV typea)
Intact
Deletion
Total
No. of isolates in each stx genotype
stx1a
stx2a
stx2c
0
3
3
21
6
27
32
0
32
stx1a/ stx2a stx1a/ stx2c stx1a/ stx2a/ stx2c stx2a / stx2c
0
105
105
14
0
14
1
0
1
21
0
21
a) Intact or 204-bp-deletion norV gene.
found that stx type alone cannot account for the variation in
hospitalization and HUS rates by clade.
In the present study, the diversity of IS629 patterns and stx
genotypes in O157 isolates collected in Yamaguchi Prefecture, Japan, were determined. The clade 8 isolates, which are
considered to show high virulence, shared a particular IS629
distribution and stx genotype. Because analysis of patterns
of IS629 distribution and stx genotype in O157 isolates can
help to determine whether or not an isolate has high virulence in humans, it is important to identify the characteristics
of O157 isolates to alert clinical laboratories to the risk of
developing severe diseases, such as HUS.
ACKNOWLEDGMENT. This work was supported in part
by a grant-in-aid for Research on Emerging and Re-emerging
Infectious Diseases (H24-Shinkou-Ippan-005) from the Ministry of Health, Labour and Welfare, Japan.
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